The overall objectives of this project are (1) a description of the proteins and complex lipids in neuronal membranes as well as the macromolecules secreted by neurons into the extracellular space, and (2) an analysis of the functional roles these molecules play in the development of neurons and the interactions neurons have with other cells, particularly during synapse formation. The system under study consists of dissociated sympathetic neurons grown in culture in the virtual absence of other cell types. Thus a relatively homogeneous population of neurons can be studied without non-neuronal contamination. These neurons can produce norepinephrine and form adrenergic synapses or they can be influenced to produce acetylcholine and form cholinergic synapses. With this transmitter decision under experimental control, it has proven possible to identify membrane-bound and secreted proteins and lipids which are transmitter- or synapse-specific; such macromolecules are produced primarily in adrenergic or cholinergic sympathetic neurons. It is proposed to produce antibodies to some of these transmitter-specific proteins. These antibodies will be used to localize the proteins on the neurons and will also be added to the cultures, both chronically and in acute incubations, and the survival, growth and differentiation of the neurons monitored. The formation and function of synapses will also be studied after antibody addition. Monoclonal antibodies are also being produced against the total surface membranes of the adrenergic and cholinergic sympathetic neurons. These antibodies will be characterized as to the specificity of their binding and the identity and localization of the antigens. The antibodies will also be added to the developing neurons in functional studies, as above. Preliminary studies have indicated that there may be striking differences in ganglioside synthesis between the adrenergic and cholinergic neurons. These differences will be further characterized and antibody production against the relevant gangliosides will be attempted.